The periodontal pathogen Porphyromans gingivalis is classified into six groups (types I-V and Ib) based on the genotype of the fimbriae A (fimA) gene. Among genotypes, fimA type II strains are thought to be most strongly related to advanced periodontitis. The present study was undertaken to develop passive immunotherapy monoclonal antibodies (MAbs) against periodontitis, which are capable of inhibiting virulency and were constructed through the immunization of outer membrane vesicles (OMV) fraction of fimAII strain, TDC60, using mouse hybridoma technology. MAbs that recognized OMV by ELISA assay were identified, and 28 clones were screened by Western blot analysis. After purifying these MAbs using protein G column, the effect of the MAb on IL-8 production from human gingival fibroblasts by OMV was examined. We selected MAb TDC4-33H, which strongly inhibited the IL-8 production with a higher MAb production rate. Since the MAb showed an individual ladder-like profile against OMV by Western blotting, we further examined the reactivity against lipopolysaccharides (LPS) from TDC60, W83 (fimAIV), and ATCC33277 (fimAI). As a result, MAb TDC4-33H recognized all LPSs. Moreover, MAb TDC4-33H significantly inhibited the LPS-stimulated IL-8 production in human gingival fibroblasts. These findings suggest that MAb TDC4-33H reacts with LPS and may be useful for passive immunotherapy through neutralizing IL-8 production in gingival fibroblasts by P. gingivalis LPS.
[Show abstract][Hide abstract] ABSTRACT: Lipopolysaccharides (LPSs) were isolated from Bacteroides gingivalis and Escherichia coli by the phenol-water and butanol-water procedures. The phenol-water-extracted LPS from B. gingivalis 381 was composed of 46% carbohydrate, 23% hexosamine, 18% fatty acid, and 5% protein. The major component sugars of this preparation were glucose, glucosamine, rhamnose, galactose, galactosamine, and mannose, and their molecular ratio was 1:0.9:0.7:0.6:0.6:0.4, respectively. Neither heptose nor 2-keto-3-deoxyoctonate was detected. The butanol-water-extracted LPS from this strain was composed of 76% glucose, 7% fatty acid, and 13% protein, and it was associated with a number of polypeptides (13 to 56 kilodaltons). The main fatty acid of both LPS preparations was palmitic acid. It was found that biological activities of LPS from B. gingivalis were comparable to those of LPS from E. coli in terms of activation of the clotting enzyme of Limulus amebocyte lysate, mitogenicity, polyclonal B cell activation, and stimulation of interleukin 1 production in BALB/c mice. Furthermore, LPS-nonresponsive C3H/HeJ spleen cells were found to yield good mitogenic responses to both phenol-water-extracted LPS and butanol-water-extracted LPS from B. gingivalis or butanol-water-extracted LPS from E. coli. On the other hand, spleen cells from LPS-responsive C3H/HeN mice responded well to all these LPS preparations.
Infection and Immunity 04/1985; 47(3):638-47. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Extracellular vesicles of Bacteroides gingivalis (type strain 33277) were isolated, and some of their biological activities were characterized. The vesicles were obtained from a 2-day culture after ammonium sulfate precipitation, differential centrifugation, and dialysis. When viewed by electron microscopy, vesicles of approximately 50 nm predominated. The results indicated that the enriched vesicle fraction had a high proteolytic activity against collagen, Azocoll, and N-alpha-benzoyl-DL-arginine p-nitroanilide. The polypeptide pattern of the vesicles was similar but not identical to that of the outer membrane. The membrane vesicles could also promote bacterial adherence between homologous cells as well as mediate attachment between two noncoaggregating bacterial species. These vesicles could thus play an important role in periodontal diseases by serving as a vehicle for toxins and various proteolytic enzymes, as well as being involved in adherence.
Infection and Immunity 02/1987; 55(1):111-7. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Three major neutrophil chemotactic peptides belonging to the interleukin-8 (IL-8) family were purified to homogeneity from the conditioned medium of the IL-1 beta-stimulated human gingival fibroblasts culture. On the basis of their molecular masses and NH2-terminal amino acid sequences, these three neutrophil chemotactic factors were identical to melanoma growth stimulatory activity-alpha (MGSA/gro-alpha), MGSA/gro-gamma and Ala-Val-Leu-Pro-Arg-IL-8 (AVLPR-IL-8), each belonging to the IL-8 family. It has been shown by the chemotaxis studies in vitro that the chemotactic activity of MGSA/gro-gamma is similar to that of MGSA/gro-alpha for both human and rat neutrophils, while AVLPR-IL-8 is chemotactic for human neutrophils but not for rat neutrophils. The in vitro findings were supported by the histological studies in vivo on the intradermal injection of either MGSA/gro-alpha or AVLPR-IL-8 into the back of the rats. The results obtained in the present experiment suggest that human gingival fibroblasts play an important role in gingival inflammation through the production of neutrophil chemotactic factors newly characterized as the IL-8 family in response to inflammatory chemical mediators including IL-1 beta.
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